A potential model system for screening methods purported to remove or disinfect TSE agents is proceeding in a collaboration between LBRUA and the Laboratory of Analytical Chemistry, DMPQ, CBER. The work seeks to confirm reports that PC12 rat-pheochromocytoma cells infected with some strains of the scrapie agent undergo marked reduction in acetyl-cholinesterase neurotransmitter activity while maintaining normal levels of adrenergic activity. Should the study succeed, PC12 cells might provide a suitable simplified assay to detect scrapie agent as a preliminary screening test of disinfectant and sterilization methods. Methods that fail to remove infectivity of scrapie agent detectable in cell culture would clearly be inadequate for practical use--where infecting doses of agent are potentially much higher than those detected by cell cultures--and would not merit further investigation in rodents. In FY 98, BSL-2 laboratory facilities and procedures suitable for the safe study of the infectious agents in cell cultures (using BSL-2 equipment and practices and elevated autoclave temperatures) were established, and basic cell-culture and fluorescent spectrophotometric and HPLC-based enzymological methods needed for the model were successfully developed. In FY 99, data from PC-12 cell cultures infected with scrapie brain analyzed by spectrophotometry demonstrated a decrease in acetycholinesterase activity compared to activity in mock-infected cells. In collaboration with Thomas Flynn, Ph.D.,CFSAN an in vitro study using primary reaggregated rat retinal cell cultures was initiated. Such cultures exhibit histotypic differentiation. Neurochemical studies have shown that the differentiated cells express the neurotransmitter enzymes that may be inhibited by scrapie and thus may provide an alternatative means of detecting scrapie infection. In FY 00, work continued on the development of an in vitro cell culture system aimed at detecting the presence of TSE agents. The project seeks to confirm reports that PC12 rat pheochromocytoma cells infected with some strains of the scrapie agent undergo a reduction in acetyl-cholinesterase (AChE)neurotransmitter activity while maintaining normal levels of adrenergic activity. Thus far, PC12 cultures inoculated with scrapie brain and analyzed for AChE activity enzyme levels by fluorescent spectrophotometry have demonstrated a decrease in AChE activity compared to the activity in mock-infected cells. However, the decrease in levels was not always consistent within the same sample group. We believe the results were confounded by the presence of minute particulates, most likely the result of incompletely digested cell membranes, which may have interfered with the spectophotometric readings. We are presently seeking to determine the most effective method for removing the particulates. The procedure is of some consequence since the AChE enzyme is associated with the cell membranes and some of the removal procedures may result in an artifactual reduction of measurable activity.. In addition, PC12 cells have been grown in a 3-dimensional(3-D)culture system which allows cultures to be fixed,paraffin embedded,sectioned and stained with appropriate antibodies for the detection of various TSE associated proteins. PC12 cells have been inoculated with either scrapie and mock-infected hamster brains and incubated for up to 28 days.The cultures have been fixed and await paraffin embedding. In FY 00 we continued a collaboration with Thomas Flynn, Ph.D., CFSAN to determine the feasibility of establishing a primary rat retinal in vitro culture system for the detection of the scrapie agent. These efforts have been extended to include primary rat brain cells grown in the 3-D culture system. Recently, using PCR techniques, we examined the mutation rate in the open reading frame of the gene encoding the prion protein (PRNP gene)in a continuous cell line under consideration as a vaccine substrate. (Mutations in the PRNP gene have been associated with familial TSEs.) The study examined changes in the PRNP gene sequences in reference HeLa cells, derived from a cervical carcinoma in 1951, and in six cell lines contaminated with HeLa cells during the 1950's and early 1960's. The lines had undergone numerous serial passages in many different laboratories during more than 30 years, suggesting that any tendency toward genetic instability in the PRNP gene might be detected. No mutation was detected, suggesting that the PrP encoding gene must have been extremely stable over hundreds of transfers in culture. Interestingly, we learned that one line of HeLa cells had lost one copy of the originally heterozygous PrP-encoding gene, most likely due to loss of one copy of chromosome 20.